Yersinia pestis, the causative agent of plague, killed more people worldwide than any other infectious disease agent. Plague is transmitted by flea bite, contact, gastrointestinal-uptake or aerosol. Y. pestis has a near global distribution in many different animal and flea hosts. Current epidemiological studies record approximately 4,000 human plague cases annually world-wide, however morbidity rates oscillate and increase slowly towards the onset of a new pandemic. The Asian Plague Pandemic (1855-1959) was halted through the live-attenuated plague vaccine EV76. Nevertheless, EV76 immunization causes serious side effects including plague disease, which preclude FDA licensure of EV76. Attenuation of Y. pestis EV76 is based on its pigmentation phenotype (?pgm), i.e. loss of yersiniabactin siderophore production. Attenuation is confined to cutaneous infection, not to bloodstream or aerosol inoculation. Individuals with hemochromatosis, whose tissue iron content is increased, are susceptible to infection with EV76 and develop plague disease. We report here that Y. pestis EV76 variants defective in both iron- (?pgm) and manganese (?yfeAB) scavenging display reduced virulence even in animals with hemochromatosis, while retaining the ability to elicit immune responses. Protective immune responses from the EV76 vaccine are focused on the F1 capsular antigen of Y. pestis and are overcome by caf1A:IS1541 escape variants. We show also that LcrV, another plague protective antigen that functions as the cap protein of Y. pestis type III machines, is glutathionylated at Cys273; this modification prevents development of protective antibody responses against LcrV during plague infection or EV76 immunization. By generating EV76 ?pgm, ?yfeAB variants with lcrVC273A, an allele for non-glutathionylated LcrV, we propose to develop live-attenuated plague vaccines that are safe, immunogenic for both F1 and LcrV and that cannot be defeated by escape mutants. This hypothesis will be tested in bubonic and pneumonic plague disease models with different animals.